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Departments of
*
Cell Signaling and
Immunobiology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304; and
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The two types of major immune responses, cellular and humoral immunity,
are differentially regulated by distinct sets of cytokines derived from
Th1 and Th2 cells; Th1 cells produce IL-2, IFN-
, and TNF-ß and
promote cellular immunity, whereas Th2 cells produce IL-4, IL-5, IL-6,
and IL-10, help in B cell functioning, and also are associated with
allergic responses (4, 5, 6, 7). Thus, a proper balance of Th1 and Th2
responses to a particular Ag or pathogen is crucial, and dysregulation
of Th1 and Th2 responses is often related to disease states (6, 7).
Evidence indicates that Th1 and Th2 cells differentiate from a common
precursor (8, 9). Furthermore, it has been shown that IL-12 and IL-4
play critical roles in Th1 and Th2 differentiation, respectively (10).
Recently, selective expression of the IL-12Rß2 subunit on Th1 cells
during Th1/Th2 differentiation has been demonstrated, and the
regulatory role of IL-4 and IFN- on this process has been shown
(11, 12, 13).
The molecular mechanisms underlying differential cytokine production in Th1 and Th2 cells remain unclear. Differences in signal transduction pathways between Th1 and Th2 cells could result in differential cytokine production. Several reports have shown that PGE2, which elevates the intracellular cAMP level, has different effects on cytokine production in Th1 and in Th2 cells, i.e., it inhibits cytokine production from Th1 cells but not from Th2 cells (14, 15). There is also an indication that the cAMP level in Th2 cells is higher than in Th1 cells (16). Other reports suggest that TCR-mediated calcium influx is selectively impaired in Th2 cells (17, 18). Selective expression of transactivators or repressors in Th1 and Th2 cells could also lead to differential cytokine production. However, the possibility of the existence of selective repressors has been refuted by a study showing that somatic cell fusion of a Th1 and a Th2 clone gives rise to a cell type that produces both Th1- and Th2-type cytokines (19).
To date, differential expression of two cytokine genes, IL-2 and IL-4,
has been shown to be regulated by differential transcriptional activity
of their promoters in Th1 and Th2 cells (20, 21). The 300-base pair
(bp)3 region of the IL-2
promoter can mediate Th1-specific expression of the gene (20, 21).
However, no cis-regulatory element within this region has
been shown to be involved in differential regulation of the IL-2 gene
in Th1 and Th2 cells. Although circumstantial evidence suggests that
differential regulation of NF-
B may be involved in Th1-specific
expression of the IL-2 gene (21), no direct evidence has been provided.
Extensive studies have focused on the IL-4 gene, and it has been shown
that the control region of Th2-specific gene activity resides within
the proximal 800-bp IL-4 promoter (21, 22). Furthermore, the region
spanning -60 to -35 has been shown to confer Th2-specific promoter
activity (20). This region has also been shown to interact with
Th2-specific transcription factor c-Maf, thereby controlling
tissue-specific expression of IL-4 (19).
On the other hand, the molecular mechanisms by which Th1 and Th2 cells differentially express the IL-5 gene remain largely unknown. Using EL-4 cells, we have found that cAMP is essential for PMA-dependent expression of the IL-5 gene, while it almost completely inhibits that of the IL-2 gene (23). cAMP exerts its differential effects on the expression of the IL-2 and IL-5 genes through the promoter regions. We have previously identified four cis-regulatory elements (designated IL-5A, IL-5P, IL-5C, and IL-5CLE0) (24) that are necessary for full activity of the IL-5 promoter in response to PMA and cAMP signals.
In this study, we examined the cis-regulatory elements and nuclear factors that account for Th2-specific expression of the IL-5 gene, using Th1 and Th2 clones as well as in vitro-generated Th1 and Th2 cells from OVA-specific TCR-transgenic mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N6,O2-dibutyryl cAMP (Bt2cAMP) was purchased from Sigma Chemical Co. (St. Louis, MO) and used at final concentrations of 1 mM. PMA, A23187, and ionomycin were purchased from Calbiochem (La Jolla, CA) and were used at final concentrations of 50 ng/ml, 0.5 µM, and 1 µM, respectively.
Th clones
Stimulation and maintenance of Th clones were as described (25). D10.G4.1 (from Dr. C. Janeway, Yale University, New Haven, CT), a conalbumin-specific Th2 clone derived from AKR/J mice, and HDK1 (26), a keyhole limpet hemocyanin-specific Th1 clone derived from BALB/c mice, were maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME, 200 U/ml mouse rIL-2, and 10% FCS. Th clones were stimulated every 2 wk with irradiated splenic APCs from mice of appropriate haplotype and specific Ags and cultured for 2 wk before preparation of RNA and nuclear extracts.
Cytokines, Abs for cytokines, and peptide
Mouse rIL-4 was from Dr. S. Menon (DNAX Research Institute). Mouse rIL-12 was obtained from PharMingen (San Diego, CA). Purified rat anti-mouse IL-4, 11B11, (27) was supplied by Dr. J. Abrams (DNAX Research Institute). Anti-IL-12 mAbs (C17.8) were as described (28). The antigenic OVA peptide from chicken OVA (OVA323–339) was synthesized on an Applied Biosystems model 430 peptide synthesizer (Foster City, CA.).
Transgenic CD4+ T cells
Preparation and culture of polarized Th1 and Th2 populations
from TCR-transgenic mice (D011.10 TCR-
ß on a BALB/c genetic
background) (29) were as previously described (30).
CD4+ T cells were stimulated weekly with OVA-peptide
(OVA323–339, 0.3 µM) presented by irradiated BALB/c
splenic APCs in the presence of IL-12 (10 ng/ml) and anti-IL-4
(11B11, 10 µg/ml), to polarize toward the Th1 phenotype, or IL-4 (10
ng/ml) and anti-IL-12 (C17.8, 10 µg/ml), to polarize toward the
Th2 phenotype.
Plasmids
pmIL5Luc(1.2), which contains the mouse IL-5 promoter (-1174 to +33) fused to the luciferase gene, as well as the promoterless control pUC00Luc have been described (23). pmoIL-2-321Luc containing the mouse IL-2 promoter (-327 to +46) fused to the luciferase gene is described in Tsuruta et al. (31), and the linker-scanning mutants of the IL-5 promoter, LS939/933, LS103/97, LS69/63, and LS57/51, are as previously described (24). The reference plasmid pRSV-LacZ in which the ß-galactosidase gene is under the control of the Rous sarcoma virus LTR was provided by Dr. A. Tsuboi (National Institute for Basic Biology, Okazaki, Japan). The plasmid mc5b8 (32), containing the murine GATA-3 in pGEM7Zf (Promega Corp., Madison, WI), was provided by Dr. D. Engel (Northwestern University, Chicago, IL). pMEGATA3 was prepared by sense insertion of the EcoRI fragment of GATA-3 cDNA from mc5b8 into the eukaryotic expression vector pME18S (33).
In vitro synthesis of GATA-3 protein
The plasmid mc5b8 was transcribed from the T7 promoter and translated in a wheat germ lysate using a TnT-Coupled Transcription/Translation Kit (Promega) according to the manufacturer’s instruction.
Transfection into Th clones and EL-4 cells
Th clones were transfected with plasmid DNA by electroporation 4 days after allogenic stimulation. The cells were washed once with serum-free RPMI 1640, then resuspended at 2.5 x 107 cells/ml. Cell suspensions (0.4 ml, 1 x 107 cells) were incubated with 10 µg of reporter constructs and 1 µg of pRSV-LacZ for 10 min at room temperature and transferred to 0.4 cm electroporation cuvettes (Bio-Rad, Richmond, CA). The cells were electrophorated using a Bio-Rad Gene Pulser at 310 V and 960 µF and allowed to recover for 10 min at room temperature. After culture for 24 h in the fresh medium, the cells were either unstimulated or stimulated with PMA and A23187 for 16 h and harvested for luciferase as well as ß-galactosidase assays.
EL-4 cells (1 x 107 cells) were transfected with 1 µg of reporter plasmid, 4 µg of activator plasmid, and 0.15 µg of pRSV-LacZ by the DEAE-dextran method as described previously (23). At 36 h after transfection, the cells were either unstimulated or stimulated with PMA and ionomycin for 12 h and assayed as above.
Antibodies
The mouse monoclonal anti-GATA-3 Ab (34) was provided by Dr. M. Yamamoto (University of Tsukuba, Tsukuba, Japan), and the GATA-4 Ab, GATA-4 (C20), a goat polyclonal Ab, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
RNA extraction and RT-PCR analysis
Total cellular RNA was isolated from T cell clones or Th1 and
Th2 cells using a RNeasy kit (Qiagen, Chatsworth, CA). One microgram of
total RNA was reverse transcribed in a 20 µl reaction, and the cDNA
samples were diluted with sterile distilled water. An aliquot of this
was used for a PCR reaction using each pair of sense and antisense
primers given in Table I
. The number of
PCR cycles and the amount of cDNA were titrated for each primer set,
and then a condition that gave an optimal signal without saturation was
chosen. PCR conditions were 94°C for 2 min, followed by: 20 to 30
cycles of 94°C, 30 s; 60 to 65°C, 30 s; 72°C, 30
s. The PCR products were separated on agarose gels, stained with CYBR
Green (Molecular Probes, Eugene, Oregon), and were then visualized
using ImageQuant (Molecular Dynamics, Sunnyvale, CA).
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Cells were treated for 2 h with various reagents, as
described in the figure legends, and nuclear extracts were made as
described (35). EMSAs were performed using the double-stranded
oligonucleotides given in Table II. Each
single-stranded oligonucleotide was purified on a denaturing
polyacrylamide gel before annealing. The annealed oligonucleotides were
32P labeled with Klenow fragment and purified on 12%
polyacrylamide gels. The DNA-binding reactions were performed at room
temperature for 30 min with 2 to 5 µg of nuclear extracts, 0.5 µg
of poly(dI-dC), 10 mM HEPES (pH 7.9), 10% glycerol, 1 mM EDTA, 1 mM
DTT, 100 mM KCl, and 0.5 ng of probe in a total volume of 10 µl. The
samples were resolved on a 5% nondenaturing polyacrylamide gel at 120
V in 0.5x Tris-borate-EDTA buffer, and the results were visualized by
autoradiography. Protein content was determined by the Bradford assay
with a kit provided by Bio-Rad.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Th1 clone, HDK1, produces IL-2 in response to a T cell activation signal, whereas the Th2 clone, D10.G4.1, produces IL-5 (26). In addition, PMA and calcium ionophore stimulation can induce IL-5 production to the same extent as anti-CD3 stimulation in several Th2 clones, such as D10.G4.1 and CDC35 (25).
To verify exclusive expression of the IL-2 gene by HDK1 cells and of
IL-5 by D10.G4.1 cells, we performed sensitive RT-PCR analysis using
RNA from unstimulated and stimulated cells (Fig. 1). The Th1 clone, HDK1, expressed IL-2
but not IL-5 in response to PMA and ionomycin stimulation, whereas the
Th2 clone, D10.G4.1, expressed IL-5 and a barely detectable amount of
IL-2. We have previously reported that cAMP activated the IL-5 gene
synergistically with PMA, whereas it suppressed PMA-induced IL-2
transcription in EL-4 cells (23). There are also other indications
suggesting involvement of the cAMP signal in IL-5 production (14, 15, 25, 36, 37). However, HDK1 (Th1) cells did not transcribe the IL-5 gene
even in the presence of cAMP, although its inhibitory effect on IL-2
gene expression could be detected. Unlike EL-4 cells in which the cAMP
signal is essential for IL-5 expression, D10.G4.1 (Th2) cells expressed
a considerable amount of IL-5 with PMA and calcium ionophore
stimulation alone, and cAMP had only an augmenting effect.
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To assess whether the Th2-specific expression of the IL-5 gene is
directly associated with the promoter activity, an IL-5
promoter-reporter gene construct was introduced into HDK1 and D10.G4.1
cells by transient transfection, as described in Materials and
Methods. An IL-2 promoter construct was also tested in parallel,
as a Th1-specific control. We previously showed that the 1.2-kb
promoter region of mouse IL-5 gene was sufficient to induce promoter
activity in EL-4 cells. When introduced into the Th2 clone D10.G4.1
cells, pmIL5Luc(1.2) containing the 1.2-kb mouse IL-5 promoter showed a
substantial inducible luciferase activity in response to PMA and
calcium ionophore A23187. In contrast, pmoIL-2-321Luc containing the
321 bp mouse IL-2 promoter showed no significant activity relative to
that of control pGL2-promoter (Promega) in D10.G4.1 cells (Fig. 2).
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IL-5C and IL-5CLE0 are critical for promoter activity in D10.G4.1 cells
The differential ability of D10.G4.1 and HDK1 cells to induce IL-5
promoter activity may result from selective expression of an essential
positive factor(s) or a repressor(s) in respective cell types. To
address this hypothesis, we investigated possible positive or negative
elements in the IL-5 promoter by mutation analysis. We had previously
defined four cis-regulatory elements—IL-5A, IL-5P, IL-5C,
and IL-5CLE0—of the IL-5 promoter responding to PMA and
Bt2cAMP in EL-4 cells (Fig. 3A). To examine whether
any of these cis elements was responsible for Th2-specific
activation of the IL-5 promoter, four luciferase reporter plasmids,
each carrying a mutation in one of the four elements, i.e., IL-5A
(LS939/933), IL-5P (LS103/97), IL-5C (LS69/63), and IL-5CLE0 (LS57/51)
(Fig. 3A), were transiently transfected into HDK1 and
D10.G4.1 cells. Constructs containing a mutation in either IL-5A or
IL-5P still retained IL-5 promoter activity, albeit at a reduced level,
whereas mutations in either IL-5C or IL-5CLE0 abrogated IL-5 promoter
activity in response to PMA and A23187 in D10.G4.1 cells (Fig. 3B). Thus, both IL-5C and IL-5CLE0 are essential for
promoter activity in D10.G4.1 cells, while the IL-5A and IL-5P are
required for optimal activity. On the other hand, none of these
constructs was activated in HDK1 cells, suggesting that
cis-acting repressor elements may not reside within these
four sites.
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The essential role of IL-5C and IL-5CLE0 for IL-5 promoter
activity suggests that in Th1 cells, the lack of IL-5 promoter activity
may be due to the absence of factors operating on these sites.
Since, in many cases, cell type-specific gene expression is controlled
by cell type-specific transcription factors, we examined whether IL-5C
and IL-5CLE0 interact with DNA-binding proteins in a Th2-specific
manner. Since no clear boundary between these two elements has been
identified, we initially used the -74 to -38 region, which includes
both IL-5C and IL-5CLE0 (74–38, Fig. 4A), as a probe in
EMSA. Nuclear extracts from D10.G4.1 cells stimulated with PMA and
ionomycin formed four principal complexes with the 74–38 probe (I, II,
III, and IV, Fig. 4B, lane 4). Comparing the
electrophoretic profile of unstimulated (lane
3) and stimulated (lane 4)
D10.G4.1 nuclear extracts, the amount of complex II and III was
increased in stimulated D10.G4.1 cells (lane
4), whereas the amount of complex I and IV was not changed
upon stimulation. In HDK1 cells, which did not express the IL-5 gene
(Fig. 1
), formation of complex I, II, and III and the inducibility of
complex II and III were also detected (lanes 1 and
2). Interestingly, however, complex IV was not
detected in HDK1 cell nuclear extracts, raising the possibility that a
component(s) of the complex is Th2 specific. Other minor complexes with
different mobility could be detected, but their appearance was variable
depending on the different nuclear extract preparations. NF-B
binding, as a control, was detected in both cell types
(lanes 5–8). Consistent with previous
reports, the ratio of the p65/50 form to p50/50 is higher in HDK1 cells
than D10.G4.1 cells (21).
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To check the sequence specificity for each complex, segments of
the sequence of the 74–38 probe were mutated and tested for their
effects on complex formation (Fig. 5A). All four complexes
were effectively inhibited by the addition of unlabeled wild-type
74–38 oligonucleotide (Fig. 5B, lane 2).
Interestingly, addition of mutIL-5C, which contains a 3-bp substitution
on the IL-5C element, inhibited complex I, II, and III but not IV
(lane 3). On the other hand, addition of
mutIL-5CLE0, which contains a 3-bp substitution on IL-5CLE0, inhibited
complex IV but not I, II, and III (lane 4).
The control NF-B oligonucleotide did not affect any of these
complexes (lane 5). Thus, it appeared that
complex I, II, and III bound to IL-5CLE0, whereas complex IV bound to
IL-5C. To further confirm this conclusion, we next used two smaller
overlapping oligonucleotides for the binding competition assay. As
expected, the IL-5C oligonucleotide (Fig. 5A, residues -74
to -54) was able to inhibit complex IV as effectively as the 74–38
oligonucleotide, but not complex I, II, and III (Fig. 5C, lane
3), whereas the IL-5CLE0 oligonucleotide (Fig. 5A, residues -61 to -36) inhibited complex I, II, and III
but not complex IV (lane 4). A direct binding
assay also showed that the IL-5C probe formed a complex with a nuclear
factor(s) (designated NFIL-5C) that migrated with mobility similar to
complex IV (lane 5), whereas the IL-5CLE0
probe formed complexes with nuclear factors (designated NFIL-5CLE0)
that migrated with mobility similar to complex I, II, and III
(lane 6). The slight difference in mobility
between the complex IV and NFIL-5C may be caused by the different
configuration of protein-DNA complexes between the probes, since a
similar difference was also observed in EMSA using in vitro-translated
GATA-3 proteins (data not shown). The difference in mobility of the
protein-DNA complex, depending on the location of the binding site on
DNA, has been demonstrated in a GATA protein (38).
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We next examined the Th1 and Th2 specificity of NFIL-5C by EMSAs
using nuclear extracts from HDK1 and D10.G4.1 cells either unstimulated
or stimulated with PMA and ionomycin. The NFIL-5C complex appeared
constitutively only in nuclear extracts from D10.G4.1 cells but not
from HDK1 cells (Fig. 6A). To confirm that
these were not artifacts instilled by long-term culture, we further
verified the Th2 specificity of NFIL-5C using freshly generated Th1 and
Th2 cells. Th1 and Th2 cells were generated in vitro from
CD4+ T cells derived from OVA-specific transgenic mice as
described in Materials and Methods. EMSAs using nuclear
extracts from Th1 and Th2 cells revealed that the appearance of the
NFIL-5C complex is restricted to Th2 cells (Fig. 6B, lanes 4
and 5) and absent from Th1 cells (lanes
2 and 3) regardless of stimulation. Sp1 binding
was detected in both cell types (lanes 6–9).
These results demonstrated that NFIL-5C is a Th2-specific factor(s) and
also strongly suggested that NFIL-5C plays a role in regulating the
expression of the IL-5 gene in a Th2 cell-specific manner.
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To identify specific bases critical for NFIL-5C complex formation,
serial 3-bp mutations were introduced in the IL-5C site (Fig. 7A) and tested for
effects on complex formation by binding competition analysis. Addition
of Cm1, Cm2, and IL-5CLE0 oligonucleotides failed to inhibit binding
(Fig. 7B, lanes 4–7 and 12), whereas
addition of Cm3 and Cm4 oligonucleotides inhibited binding as
effectively as the wild-type IL-5C oligonucleotide (Fig. 7B,
compare lanes 2 and 3 with lanes
8–11). Similarly, the oligonucleotide containing the
mutations at position -73 to -71 inhibited the binding (data not
shown). These results indicate that residues -69 through -63 were
essential for NFIL-5C binding. It is important to note that these
sequences contain overlapping binding sites for GATA proteins (Fig. 7A).
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GATA) containing a
GATA-3 binding site from the human T cell receptor -chain gene
enhancer (39). NFIL-5C complex formation was inhibited by addition of
excess TGATA as well as IL-5C oligonucleotides (Fig. 8A, lanes 2 and
3), but not by the IL-5CLE0 oligonucleotide
(lane 4). Conversely, the unlabeled IL-5C
oligonucleotide was able to inhibit the formation of the DNA-protein
complex when the labeled TGATA probe was used
(lane 7).
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B). The NFIL-5C complex from both
D10.G4.1 and in vitro-generated Th2 cell nuclear extracts was
supershifted by an Ab specific for GATA-3 (34) (lanes
2 and 5), but not by an Ab raised against GATA-4
(lanes 3 and 6). Another GATA-4
antiserum that does not cross-react with GATA-1, -2, or -3 (42) did not
supershift the NFIL-5C complex (data not shown). Similar results were
obtained with the TGATA probe (data not shown). These Abs did not
supershift the NF-B complexes (lanes 8 and
9). Differential expression of GATA-3 in Th1 and Th2 cells
Supershift EMSA results indicated that the Th2-specific nuclear
factor NFIL-5C is GATA-3 or a related protein(s). Thus, we next
examined whether GATA-3 is differentially expressed in Th1 and Th2
cells. RT-PCR analysis was performed using RNA from unstimulated and
stimulated Th1 and Th2 clones as well as in vitro-generated
TCR-transgenic Th1 and Th2 cells (Fig. 9A). GATA-3 expression
was detected only in the Th2 clone, D10.G4.1 cells, but not in the Th1
clone, HDK1 cells. To compare the level of GATA-3 transcripts in HDK1
and D10.G4.1 cells, we titrated the amount of cDNA in PCR reactions. As
shown in Figure 9B, D10.G4.1 cells express a significantly
higher level of GATA-3 than HDK1 cells, while both cells express a
comparable level of HPRT transcripts. This differential expression of
GATA-3 was also observed in Ag-specific Th2 and Th1 cells obtained from
the TCR-transgenic mice (Fig. 9A). These data are in
keeping with recent findings from Zheng and Flavell (43). In contrast,
IL-12Rß2 transcripts were detected in HDK1 and Th1 cells but not in
D10.G4.1 and Th2 cells, as previously reported (12, 13). As expected,
IL-5 expression was restricted to D10.G4.1 and Th2 cells.
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We next asked whether GATA-3 directly regulates IL-5 promoter
activity through the IL-5C element. First, we determined whether
recombinant GATA-3 can bind to IL-5C. We prepared murine GATA-3
proteins by in vitro transcription and translation using a wheat germ
system and tested their ability to bind IL-5C. The IL-5C probe formed a
DNA-protein complex with wheat germ extracts from in vitro
transcription and translation reactions programmed with recombinant
GATA-3, but not with those programmed with the control vector (Fig. 10A, lanes 1 and
2). The complex formation was inhibited by addition
of the wild-type (WT) IL-5C oligonucleotide (lane
3) but not by the Cm1 oligonucleotide (lane
4), which failed to bind NFIL-5C (Fig. 7B). Thus, we concluded that GATA-3 binds to the
IL-5C site in a sequence-specific manner.
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B). The transactivation of the IL-5 promoter by
GATA-3 required the IL-5C element, since GATA-3 did not transactivate
LS69/63, which contains a mutation in IL-5C. Taken together with the
results from the in vitro binding assay, these result suggest that
GATA-3 regulates the IL-5 promoter by direct interaction with the IL-5C
site, but is not sufficient by itself to activate the IL-5
promoter. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we provide strong evidence that Th2-specific expression of the IL-5 gene is controlled by the interaction of a Th2-specific transcription factor with the critical cis element of the gene. Using Th1 clone HDK1 and Th2 clone D10.G4.1 as a model system, we explored the molecular mechanisms underlying differential expression of the IL-5 gene in Th1 and Th2 cells. First, we confirmed, by sensitive RT-PCR assays, that induction of IL-5 gene transcription was restricted to D10.G4.1 cells and that cell type-specific expression of the IL-5 gene was directly associated with the inducibility of the promoter in D10.G4.1 cells but not HDK1 cells. We further demonstrated that IL-5C and IL-5CLE0 were critical for the function of the IL-5 promoter in D10.G4.1 cells. Moreover, IL-5C interacts with the D10.G4.1 cell-specific nuclear factor (NFIL-5C), which is related to GATA-3. Finally, we show that GATA-3 is preferentially expressed in D10.G4.1 (Th2) cells. Importantly, these findings were not limited to HDK1 and D10.G4.1 cells. We further confirmed our results using freshly generated TCR-transgenic Th1 and Th2 cells (30), which strongly suggests that this is not a phenomenon restricted to Th clones carried long-term in vitro.
Sequence similarity between the binding sequence of NFIL-5C and GATA
protein consensus and cross-competition for binding between IL-5C and
TGATA suggest that the NFIL-5C complex contains GATA proteins.
Involvement of GATA proteins in IL-5 gene transcription through IL-5C
has also been shown in other systems using tumor cell lines (40, 41).
GATA-3 seems to be the major component of the NFIL-5C complex, since
most of the complex was supershifted by anti-GATA-3 Ab. This notion
is in good agreement with our RT-PCR results showing that the
expression profile of GATA-3 in these cells was closely correlated with
the appearance of the NFIL-5C complex. Indeed, recombinant GATA-3 could
bind to IL-5C specifically and activated the IL-5 promoter via the
IL-5C element. These results, together with the overlapping expression
profile of the GATA-3 and the IL-5 genes, suggest that GATA-3 may
control Th2-specific expression of the IL-5 gene through IL-5C. GATA
proteins are a group of transcription factors with a C4 zinc finger
DNA-binding domain that recognizes the consensus sequence A/TGATAG/A as
well as some related sequences (46). GATA-3 is preferentially expressed
in the T cell hemopoietic lineage and plays an important role in T cell
development (47). Functional GATA sites have been identified on the
enhancers of several T cell-specific genes including the TCR
(48).
Our results are in agreement with the recent report by Zheng and Flavell that identified GATA-3 as a gene selectively expressed in Th2 cells by cDNA subtraction between in vitro-generated Th1 and Th2 cells (43). Their antisense as well as transgenic results suggest clear involvement of GATA-3 in the expression of Th2 cytokine genes such as IL-4, IL-6, IL-10, and IL-13, but the effect of a reduced level of GATA-3 on IL-5 expression was minimal in the antisense GATA-3-expressing D10 cells. However, our results suggest that IL-5C is absolutely required for the IL-5 promoter activity in D10 cells. The results from Zheng and Flavell (43) indicate that the difference in the level of GATA-3 in the control D10 cells and the antisense GATA-3-expressing D10 cells is not as dramatic as that seen in Th1 and Th2 clones. Their results also clearly showed increased expression of the IL-5 gene, albeit to a lesser extent than IL-4, IL-6, or IL-10, in IL-12-driven Th1 cells obtained from GATA-3-transgenic mice. Zheng and Flavell (43) also demonstrated that TCR expression levels were not altered by a reduced expression of GATA-3 and that the extent of inhibition was different among Th2 cytokines. Thus, the GATA site of each of these genes may have a different affinity for GATA-3, and the range of the GATA sites being occupied may depend on the level of GATA-3 expression. In this respect, it is interesting to note that the IL-5C sequence comprises the inverted overlapping GATA-3-binding sites with high affinity (3' part) and intermediate affinity (5' part) (49). It has been reported that double GATA sites often have higher affinity for GATA proteins (50). In addition, chromatin accessibility and interaction with other factors could also affect preferential association of GATA-3 with each target site in vivo. Alternatively, the NFIL-5C complex may consist of other factors closely related but not identical to GATA-3. Further in vivo and in vitro experiments will be required to clarify this question.
Mutational analysis indicated that not only IL-5C but also IL-5CLE0 was indispensable for IL-5 promoter activity. In contrast to NFIL-5C, NFIL-5CLE0 was induced by stimulation in both HDK1 and D10.G4.1 cells. However, we cannot rule out the possibility that the components and/or modification status of the NFIL-5CLE0 complex may not be exactly the same between Th1 and Th2 cells, resulting in differential IL-5 gene transcription. Interestingly, Naora et al. reported that the oligonucleotide containing IL-5CLE0 interacted with an inducible nuclear factor present in D10.G4.1 cells but not in HDK1 cells, using their specific stimulation and binding conditions (51). Thus, it may be possible to distinguish the Th2-specific component(s), if any, within the NFIL-5CLE0 complex under the conditions they used. Further biochemical analysis will be required to clarify the components of NFIL-5CLE0 and its role in Th2-specific expression of IL-5.
Since both IL-5C and IL-5CLE0 are essential for the functioning of the
IL-5 promoter, cells that have both NFIL-5C and NFIL-5CLE0 can support
promoter activation (Fig. 11).
Unstimulated Th1 cells in which neither IL-5C nor IL-5CLE0 is occupied
are unable to induce IL-5 promoter activity. Upon stimulation,
NFIL-5CLE0 complex formation is induced, but is still insufficient for
IL-5 promoter activation. Similarly, occupation of IL-5C by NFIL-5C in
unstimulated Th2 cells is not sufficient for promoter activity. Further
occupation of IL-5CLE0 by NFIL-5CLE0 after stimulation is needed to
trigger promoter activation. This notion is in good agreement with the
expression profile of the GATA-3 and IL-5 as well as the result from
cotransfection of GATA-3, indicating that both GATA-3 and
stimulation-dependent factors are required to activate the IL-5 gene.
Currently, we do not know how NFIL-5C and NFIL-5CLE0 cooperate to
activate the IL-5 promoter. It is possible that binding of both sites
may trigger the recruitment of additional factors, such as coactivator
and/or transcription initiation machinery, to turn on the IL-5
promoter. Further studies will be required to address this
hypothesis.
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Taken together, our studies dissect the critical regulatory mechanisms for IL-5 expression, which may provide not only an insight into the pathogenesis of IL-5-associated allergic diseases but also the potential for alternative approaches to therapy. Furthermore, this system will provide a useful tool for studying the mechanisms for differential regulation of cytokine genes in Th1 and Th2 cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Naoko Arai, Department of Cell Signaling, DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304.
3 Abbreviations used in this paper: bp, base pair(s); EMSA, electrophoretic mobility shift assay; kb, kilobase; Bt2cAMP, N6,O2-dibutyryl cAMP; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
Received for publication July 10, 1997. Accepted for publication November 6, 1997.
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